Enhancement of Influenza Viral Yields by Engineered Embryonated Eggs and Cell Lines (I)

Egg-based propagation of virus for influenza vaccine has been used for more than 60 years. At the moment for writing this grant proposal, almost all marketed influenza vaccines are grown in embryonated eggs. Egg-based vaccines have been considered to be relative safe. However, the yields for human strains sometimes could not reach a satisfactory amount. Thus, there is a need to increase viral yields in eggs. We found that hSLU7 significantly enhanced viral production in avian cells. It is because that the human splicing factor, hSLU7, can enhance viral production through increasing the level of M2 ion channel protein that is translated from an alternative spliced M mRNA. Therefore, this proposal would like to deliver human SLU7 gene (hSLU7) into chicken embryonated eggs to increase viral yields for vaccine production. On the other hand, we would like to knock down a species specific inhibitor to promote avian influenza viral yields in mammalian cells. We have identified that elongation factor Tu mitochondria (TUFM) is a mammalian cell specific inhibitor for avian influenza virus replication. Our idea is to knock down TUFM in MDCK or Vero cells for a better avian influenza vaccine production. This study would be also a pioneer approach of a “proof-of-concept” study that transferring or knocking down a host specific factor into production system would promote vaccine yields. Three specific aims of the proposal were summarized: 1. Continue to engineer hSLU7 transgenic embryonated eggs Previopusly we have sucessfully used the lentivirus to deliver hSLU7 into chicken embryonated eggs. However, we found that letivirus infection itself inhibited influenza virus replication in embryonated eggs. In the current proposal, we would like to use Tol2 system to achieve this aim. A plasmid containing Tol2 transposase and a plasmid containing hSLU7 gene with Tol2 transposable elements will be delivered into embryonated eggs by electroporation. 2. To evaluate the viral yields in hSLU7-transferred embryonated eggs (hSLU7-eggs) and analyze the composition of the vaccines that produced by hSLU7-eggs Viral titer will be determined by plaque assay. Moreover, haemagglutinin (HA) antigen will be quantified by a single radial immune-diffusion (SRID) assay. Seasonal influenza vaccines are usually trivalent. Each dose would contain 15 g of each two influenza A subtypes (e.g. H1N1 and H3N2) and 15 g of one influenza B strain. Therefore, H1N1, H3N2 and influenza B viruses will be inoculated into hSLU7-eggs and the HA antigens for each virus will be analyzed. 3. To engineer cell lines that can promote influenza vaccine productions In general, avian influenza virus cannot replicate efficiently in mammalian cell lines because of host species restriction. Knockdown of host restriction factor would promote avian flu vaccine production in mammalian cell lines. TUFM, a host restriction factor that only exists in mammalian cells, not in avian cells, will be knocked down by siRNA and the stable cell line will be established, MDCK and Vero cells will be used in this study because they have been used in vaccine industry.

Project ID:PC10301-0035
External Project ID:MOST103-2321-B182-009